*Purpose: Chronic rejection (CR) results from the development of immunosuppressant-resistant allograft vasculopathy and fibrosis and remains a leading cause of recipient death one-year post-heart transplant (HTx). The blockage of graft vessels and progressive graft scarring causing CR may reflect failed tissue repair. Recent studies have suggested not only suppressive but important reparative functions of regulatory T cells (Treg) after tissue injury. Yet, recipient alloimmune responses to persistent MHC differences will cause sustained injury that could impact on repair responses. Thus, we test the hypothesis that Treg repair responses in the HTx become dysregulated due to a sustained inflammatory response by alloreactive immune cells.

*Methods: B6 (H-2^b) CD45.1⁺ mice received heterotopic HTx from Bm12 (H2-I-A^bm12) heart transplants that were either deficient or competent for the damage-associated molecular pattern (DAMP) IL-33 (n=4). At day (d) 14 post-HTx, single-cell RNAseq and CITE-seq analysis was used to compare recipient leukocytes isolated from HTx digests. B6 Foxp3^YFP-CrexAreg^fl/fl and Foxp3^YFP-Cre mice also received heterotopic Bm12 (H2-Ab1^bm12) heart transplants. At day 100 post-HTx, isolated grafts were asessed for CR. The impact of Treg and Areg on fibroblast proliferation, survival, and migration was assessed ex vivo using an IncuCyte Live-Cell Imaging Systems. Naïve T cells (controls), as well as Tregs stimulated for 2 or 3 weeks with IL-2 alone or with IL-33 were assessed for methylation percentages at CpG sites of various Treg-associated genes.

*Results: Fibroblasts are the main source of IL-33 in the HTx and >800 genes were significantly modulated in HTx-infiltrating Treg by local IL-33 at d14. This included the reparative growth factor amphiregulin (Areg; 2.25x; P=0.0241). Areg is has been implicated in Treg-mediated early repair of muscle and lung epithelium. Yet, the specific deletion of Areg from Treg provided protection against HTx fibrosis at d100. This was consistent with our observation that Areg drives the migration and activation of fibroblasts into wound sites in vitro. Sustained exposure to IL-33 also increased methylation of Foxp3 and other genes, suggesting that sustained DAMP stimulation makes Treg unstable.

*Conclusions: In total, our studies identify how a dysregulated repair response involving interactions between IL-33⁺ fibroblasts and recipient Treg contributes to the progression of HTx CR.

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